Currently, the sole cure for both acute and chronic liver disease and liver failure is transplantation. However, this treatment is restricted by a lack of donor organs. In the US alone, 17,000 patients are on the transplant waiting list. Many of them die whilst waiting for a transplant. There is therefore an urgent need for a device which can temporarily perform the function of a patient's liver, keeping them alive whilst a suitable donor organ is found, or which provides an environment which ensures the patient does not die whilst the patient's own liver recovers sufficient functionality for the patient's survival.
There are two principal types of liver machines:                A purely artificial machine; and        A bio-artificial machine.        
Both rely on perfusion of a patient's plasma or blood into an extracorporeal circuit for a period of 6 h or more.
Purely artificial systems exist and a number of bio-artificial systems are in development.
Purely artificial systems, such as albumin dialysis, are however unable to replace all the liver functions including:                Detoxification,        Biotransformation,        Synthesis, and        Storageand whilst they have proved relatively safe in clinical trials they have not given rise to a significant improvement in patient survival.        
The purely artificial systems are solely physical/chemical in nature, and provide a detoxification function by adsorption/exchange on e.g. resin, charcoal, ion exchange columns or albumin, or combinations of these.
In contrast the bio-artificial livers (BALs) contain a biological component, i.e. liver cells, either alone, or in conjunction with an artificial device as a hybrid system. The hypothesis underlying the incorporation of liver cells is that liver function is so complex, comprising multiple synthetic, detoxification, and metabolic pathways, that crude mechanical devices will always be inadequate to replace the range of function desired; furthermore the functions critical to buying time for liver function to recover, have not been fully defined and the use of liver cells allows both defined and undefined functions to be replaced. For the biological component, isolated liver cells or occasionally liver slices are used, and systems have used either human or animal (most often porcine) cells.
The majority of early BALs used hollow fibre cartridges in which cells were separated from plasma or whole blood by a membrane. Pore sizes of the membrane differed between systems, some limited to transfer of molecules <10,000 daltons, some with pore sizes as large as 2 micron.
More recently other configurations have emerged which better address mass transfer limitations. They include:                An AMC-BAL which contains cells attached to a polyester matrix which is exposed directly to oxygenated plasma, (Flendrig L M, LaSoe J W, Jorning G G A, Steenbeek A, Karlsen O T, Bovee W M M J, Ladiges N C J J, TeVelde A A, Chamuleau R A F M. In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound non-woven polyester matrix for hepatocyte culture as small aggregates. Journal of Hepatology 1998; 26: 1379-1392.)        A non-woven fabric bioreactor, (Li L J, Du W B, Zhang Y M, Li J, Pan X P, Chen J J, Cao H C, Chen Y, Chen Y M. Evaluation of a bioartificial liver based on a non-woven fabric bioreactor with porcine hepatocytes in pigs. Journal of Hepatology 2006; 44: 317-324.)        A radial flow bioreactor, (Morsiani E, Brogli M, Galavotti D, Bellini T, Ricci D, Pazzi P, Puviani A C. Long-term expression of highly differentiated functions by isolated porcine hepatocytes perfused in a radial-flow bioreactor. Artif. Organs 2001; 25: 740-748.) and        The Innsbruck Bioartificial Liver containing hepatocyte aggregates. (Hochleitner B, Hengster P, Duo L, Bucher H, Klima G, Margreiter R. A novel bio-artificial liver with culture of porcine hepatocyte aggregates under simulated microgravity. Artif. Organs 2005; 29: 58-66.)The above examples all use animal hepatocytes.        
Examples of reactors with human cells include those using:                Primary hepatocytes, (Gerlach J C, Mutig K, Sauer I M, Schrade P, Efimova E, Mieder T, Naumann G, Grunwald A, Pless G, Mas A, Bachmann S, Neuhaus P, Zeilinger K. Use of primary human liver cells originating from discarded grafts in a bioreactor for liver support therapy and the prospects of culturing adult liver stem cells in bioreactors: a morphologic study. Transplantation 2003; 76: 781-786.)        Hollow fibre cartridges using well-differentiated tumour-derived cell lines such as C3A cells, (Ellis A J, Hughes R D, Wendon J A, Dunne J, Langley P G, Kelly J H, Gislason G T, Sussman N L, Williams R. Pilot-controlled trial of the extracorporeal liver assist device in acute liver failure. Hepatology 1996; 24: 1446-1451.) and        A fluidised bed bioreactor with human C3A cells encapsulated at high (about 1 million cells/ml) density into alginate. (David B, Dufresne M, Nagel M D, Legallais C. In vitro assessment of encapsulated C3A hepatocytes functions in a fluidized bed bioreactor. Biotechnol. Prog. 2004; 20: 1204-1212.)        
Various groups around the world are working with different biological components including:                The use of primary cultures of human hepatocytes;        The use of primary cultures of pig hepatocytes; and        The use of C3A cells—a proliferating cell line initially derived from a well developed human liver cell tumour.        
There are fundamental differences between any system which uses proliferating cell lines and those which use primary cells. Proliferating cell lines can be seeded singly and multiply, in situ, to form cohesive spheroids over a period of time, dependent on the doubling time of a specific cell type. In contrast, primary cells even if seeded at a very high cell density will not necessarily form close cell to cell contacts and therefore will not necessarily give rise to a true 3-dimensional environment, which is associated with up-regulation of function, as it mimics the in vivo situation.
The applicant's approach has been to use a cell line, and has similarities with the C3A approach which has not proved effective in clinical trials. However, the applicant's cell line is different and has some different functional properties. There are also fundamental differences between the housing and initial culture of the cells prior to use.
Previously, C3A cells have been used either in:                “Hollow fibre cartridge culture configuration”, or        “Uncultured”, in a fluidised bed in low occupancy alginate beads.This is in contrast to the methodology used by the applicant, who uses an uncoated alginate matrix in a fluidised bed bioreactor configuration with pre-culture of encapsulated cells to performance competence.        
The applicant's biological component, which comprises human hepatocyte cell lines cultured in a 3-D configuration, has been demonstrated, at lab scale, to provide functional liver capacity on a per cell basis, which approaches that seen in vivo for several of the liver's key functions including:                Clotting factor synthesis;        Steroid metabolism; and        Specified detoxification functions.        
Fuller details on the expression of hepatocyte-specific function, pioneered by the applicant, are given below:
Applicant has pioneered (on a laboratory scale) the culture of human hepatocyte-derived cell lines as 3-dimensional (3-D) spheroid colonies in alginate beads, as disclosed in:                Selden C, Shariat A, McCloskey P, Ryder T, Roberts E, Hodgson H.        
Three-dimensional in vitro cell culture leads to a marked upregulation of cell function in human hepatocyte cell lines—an important tool for the development of a bioartificial liver machine. Annals Of The New York Academy Of Science 1999; 875: 353-363;                McCloskey P, Edwards R J1 Tootle R, Selden C, Roberts E, Hodgson H J. Resistance of three immortalized human hepatocyte cell lines to acetaminophen and N-acetyl-p-benzoquinoneimine toxicity. J Hepatol 1999; 31: 841-851;        Selden C, Khalil M, Hodgson H. Three dimensional culture upregulates extracellular matrix protein expression in human liver cell lines-a step towards mimicking the liver in vivo? Int J Artif Organs 2000; 23: 774-781;        Khalil M, Shariat-Panahi A, Tootle R, Ryder T, McCloskey P, Roberts E, Hodgson H, Selden C. Human hepatocyte cell lines proliferating as cohesive spheroid colonies in alginate markedly upregulate both synthetic and detoxificatory liver function. Journal Of Hepatology 2001; 34: 68-77;        McCloskey P, Tootle R, Selden C, Larsen F1 Roberts E, Hodgson H J. Modulation of hepatocyte function in an immortalized human hepatocyte cell line following exposure to liver-failure plasma. Artif. Organs 2002; 26: 340-348; and        Coward S M, Selden C, Mantalaris A, Hodgson H J. Proliferation rates of HepG2 cells encapsulated in alginate are increased in a microgravity environment compared with static cultures. Artif. Organs 2005; 29: 152-158.Each of these documents are incorporated by reference.        
The advantages of this system are:                Cells proliferating in this milieu maintain a near-cuboidal cell architecture;        They have close cell-to-cell and cell-matrix organisation; and        They secrete extracellular matrix proteins and a large repertoire of liver specific secreted proteins as exemplified by:        Albumin,        Prothrombin,        Fibrinogen,        Alpha-1-antitrypsin, and        Alpha-1-acid glycoprotein.        
They express many functions at levels equivalent to those of hepatocytes in vivo, e.g. steroid metabolism, glycogen synthesis etc.
The applicant has also shown that some functions are poorly expressed or missing, but can be supplemented. For example they have demonstrated that although HepG2 clones, including the C3A subclone which is the basis of one bio-artificial device, produce urea, this is via a urea-cycle independent mechanism that does not detoxify ammonia. Thus, unmodified, such cells are unlikely to be beneficial in treating the ammonia-dependent encephalopathy of liver failure. Using gene transfer to replace two missing enzymes they have demonstrated restoration of urea production from ammonia in their HepG2 clones. (Mavri-Damelin D, Eaton S, Damelin L H, Rees M, Hodgson H J, Selden C. Ornithine transcarbamylase and arginase I deficiency are responsible for diminished urea cycle function in the human hepatoblastoma cell line HepG2. Int. J Biochem. Cell Biol. 2006.
They have also characterised them extensively with respect to liver specific function as disclosed in:                (Khalil M, Shariat-Panahi A, Tootle R, Ryder T, McCloskey P, Roberts E, Hodgson H, Selden C. Human hepatocyte cell lines proliferating as cohesive spheroid colonies in alginate markedly up-regulate both synthetic and detoxificatory liver function. Journal Of Hepatology 2001; 34: 68-77;        Selden C, Shariat A, McCloskey P, Ryder T, Roberts E, Hodgson H. Three-dimensional in vitro cell culture leads to a marked upregulation of cell function in human hepatocyte cell lines—an important tool for the development of a bioartificial liver machine. Annals Of The New York Academy Of Science 1999; 875: 353-363; and        Selden C, Khalil M, Hodgson H. Three dimensional culture upregulates extracellular matrix protein expression in human liver cell lines—a step towards mimicking the liver in vivo? Int J Artif Organs 2000; 23: 774-781)        
(All referred to previously) and                L H Damelin, M Kirwan, S Coward, P Collins, I J Cox, C Selden, H J F Hodgson. Fat-loaded insulin resistant HepG2 cells are resistant to cytokine and pro-oxidant induced damage, but become damage susceptible after down-regulation of AMP-activated kinase. BASL 2005; and        Selden C, Roberts E, Stamp G, Parker K, Winlove P, Ryder T, Platt H, Hodgson H. Comparison of three solid phase supports for promoting three-dimensional growth and function of human liver cell lines. Artif. Organs 1998; 22: 308-319.        
(Also, incorporated by reference hereto) Additionally, in an animal model of fulminant liver failure, they have shown them to exhibit an improvement in clinical and biochemical parameters.                Rahman T M, Selden C, Khalil M, Diakanov I, Hodgson H J. Alginate-encapsulated human hepatoblastoma cells in an extracorporeal perfusion system improve some systemic parameters of liver failure in a xenogeneic model. Artif. Organs 2004; 28: 476-482; and        Rahman T M, Selden A C, Hodgson H J. A novel model of acetaminophen-induced acute hepatic failure in rabbits. J Surg. Res. 2002; 106: 264-272.(Also, incorporated by reference hereto)        
Furthermore, they have demonstrated improved per bead performance by culture in a rotating cell culture system (RCCS) under simulated microgravity conditions:                Coward S M, Selden C, Mantalaris A, Hodgson H J. Proliferation rates of HepG2 cells encapsulated in alginate are increased in a microgravity environment compared with static cultures. Artif. Organs 2005; 29: 152-158 (Referred to previously); and        Human liver cells in a pilot scale fluidised bed bioreactor maintain performance in human liver failure plasma, making them suitable for a bioartificial liver. Presented at World Congress of Biomechanics, Jul. 29-Aug. 4 2006 in Munich, Germany.        
They have also tested the performance of this system in normal human plasma and plasma collected from patients with acute liver failure establishing that there is maintained viability and functional performance over 8 hours.                S. M. Coward, C. Legallais, M. Thomas, F. Tofteng, F. Larsen, H. J. Hodgson, C. Selden. Alginate-encapsulated Hepg2 cells in a pilot-scale fluidised bed bioreactor maintain performance in human liver failure plasma making them suitable for use in a bioartificial liver.Journal of Hepatology 44 [Suppl 2], S53. 2006.(Also, incorporated by reference hereto)        
However, the scale up of the biological component, from a laboratory scale size, involving no more than a 70 ml volume of alginate beads, provides significant challenges, some of which are addressed herein, the solutions to which may form the basis of independent claims.
Thus, for example, the biological component of the extracorporeal system should be:                Prepared to appropriate good manufacturing practice (GMP),        Readily transportable to centres where the patients will be hospitalised,        Conveniently packaged for storage, transport and charging a perfusion system;        Movable from the “cell-factory” through to the “clinic”, for use in extracorporeal circulation.        
U.S. Pat. No. 6,218,182 teaches a tissue engineering bioreactor for growing three dimensional tissue in which cells are seeded onto a mesh. After the tissue has been grown in the bioreactor, it is suggested that it can be frozen and preserved in the bioreactor container itself.
An aim of the present invention was to develop a chamber, sized for human use, into which the applicant could incorporate their, or another, biological component to form a bio-artificial liver that could benefit patients, but which may additionally be used to mimic a liver in drug metabolism and liver toxicity studies. In the latter case there would not necessarily be a need to cryo-preserve the cells.
This object is achieved by having a chamber, in which a biological component can be housed to form, for example, a bio-artificial liver (BAL), which is functionally modular in that it can retain the biological component in a manner which allows it to:                Proliferate the biological component (in situ);        Cryopreserve (both freeze and defrost) the biological component (in situ); and        Be perfused.        